Carrier-free biologically-active protein nanostructures

ABSTRACT

The present disclosure provides compositions and methods for efficient and effective protein delivery in vitro and in vivo. In some aspects, proteins are reversibly crosslinked to each other and/or modified with functional groups and protected from protease degradation by a polymer-based or silica-based nanoshell.

RELATED APPLICATION

This application is a continuation of U.S. patent application Ser. No.15/470,169, filed Mar. 27, 2017, now pending, which is a continuation ofU.S. patent application Ser. No. 14/498,386, filed Sep. 26, 2014, nowU.S. Pat. No. 9,603,944, which claims the benefit under 35 U.S.C. §119(e) of U.S. provisional application No. 61/883,503, filed Sep. 27,2013. The entire contents of each of the foregoing applications areincorporated herein in their entireties.

FIELD OF THE INVENTION

The present disclosure relates, in some embodiments, to the delivery ofcarrier-free, biologically-active therapeutic proteins to tissues andcells.

BACKGROUND OF THE INVENTION

Protein therapeutics, such as antibodies, cytokines, growth factors andvaccines, are important therapeutics for the treatment of a variety ofdiseases including, for example, cancer, diabetes and cardiovasculardiseases. This class of protein therapeutics has been developed rapidlyin the global pharmaceutical industry over the last few years. Proteintherapeutics have the advantages of high specificity and potencyrelative to small molecule drugs. Nonetheless, the use of proteintherapeutics is limited as a result of their intrinsic instability,immunogenicity and short half-life.

To address these limitations, there are generally two approaches: one isgenetic fusion of the therapeutic protein, and the other is use ofengineered carriers to deliver protein therapeutics. With engineeredcarriers, proteins are loaded by either encapsulation/adsorption orconjugation. Encapsulation or adsorption of proteins in/onto liposomesor nanoparticles is typically inefficient. Conjugation of proteinstypically reduces their bioactivity. Thus, both approaches areproblematic.

SUMMARY OF THE INVENTION

The present disclosure provides, inter alia, methods and compositionsfor efficient delivery of bioactive (e.g., fully bioactive) proteins.Various aspects provided herein are based, at least in part, onsurprising results showing that proteins (e.g., therapeutic proteins),reversibly and covalently crosslinked to each other through a degradablelinker can be delivered in vivo without a carrier (e.g., without albuminor other carrier) as bioactive proteins. Various other aspects describedherein are based, at least in part, on surprising results showing thatproteins, reversibly modified with functional groups and furtherprotected from degradation by a polymer-based nanoshell, can bedelivered in vivo as intact, fully bioactive proteins. Using methodsprovided herein, proteins can be incorporated into a delivery systemwith a high incorporation efficiency (e.g., greater than ˜90%) and withhigh protein drug loading efficiency (e.g., greater than ˜80%). Theseefficiencies are far higher than what has been achieved in the past.

Some aspects of the present disclosure provide compositions comprising amonodispersed plurality of carrier-free, biologically-activeprotein-polymer nanogels, wherein proteins of the nanogels arereversibly and covalently crosslinked to each other through a degradablelinker, and wherein proteins of the nanogels are crosslinked to apolymer. In some embodiments, the polymer is crosslinked to the surfaceof a nanogel (and, thus, is considered to be surface-conjugated—see,e.g., FIG. 9A).

In some embodiments, a nanostructure (e.g., nanogel) comprises, consistsof, or consists essentially of (a) one or more biologically-activeproteins reversibly and covalently crosslinked to each other through adegradable linker (e.g., disulfide linker) and (b) polymers crosslinkedto surface-exposed proteins of the nanogel (e.g., reversibly andcovalently crosslinked through a degradable linker). In someembodiments, the weight percentage of proteins crosslinked to each otheris greater than 75% w/w (e.g., greater than 80%, 85% or 90% w/w) of thenanogel.

A plurality of nanogels is considered to be “monodispersed” in acomposition (e.g., an aqueous or otherwise liquid composition) if thenanogels have the same size (e.g., diameter) relative to each other.Nanogels of a plurality may be considered to have the same size relativeto each other if the sizes among the nanogels in the plurality vary byno more than 5%-10%. In some embodiments, nanogels of a plurality areconsidered to have the same size relative to each other if the sizesamong the nanogels in the plurality vary by no more than 5%, 6%, 7%, 8%,9% or 10%. In some embodiments, nanogels of a plurality are consideredto have the same size relative to each other if the sizes among thenanogels in the plurality vary by less than 5% (e.g., 4%, 3%, 2% or 1%)

Other aspects of the present disclosure provide nanogels comprising apolymer and at least 75% w/w of proteins that are reversibly andcovalently crosslinked to each other through a degradable linker. Insome embodiments, the degradable linker is a redox responsive linker,such as, for example, a disulfide linker (e.g., Formula I).

Yet other aspects of the present disclosure provide methods of producinga plurality of carrier-free, biologically-active protein nanogels, themethods comprising (a) contacting a protein with a degradable linker(e.g., a disulfide linker) under conditions that permit reversiblecovalent crosslinking of proteins to each other through the degradablelinker, thereby producing a plurality of protein nanogels, and (b)contacting the protein nanogels with a polymer (e.g., polyethyleneglycol) under conditions that permit crosslinking of the polymer toproteins of the protein nanogels, thereby producing a plurality ofcarrier-free, biologically-active protein-polymer nanogels.

In some embodiments, the conditions of (a) include contacting theprotein with the degradable linker in an aqueous buffer at a temperatureof 4° C. to 25° C. In some embodiments, the conditions of (a) includecontacting the protein with the degradable linker in an aqueous bufferfor 30 minutes to one hour. In some embodiments, the conditions of (b)include contacting the protein nanogels with the polymer in an aqueousbuffer at a temperature of 4° C. to 25° C. In some embodiments, theconditions of (b) include contacting the protein nanogels with thepolymer in an aqueous buffer for 30 minutes to one hour. In someembodiments, the aqueous buffer comprises phosphate buffered saline(PBS).

In some embodiments, the conditions of (a) do not include contacting theprotein with the degradable linker at a temperature of greater than 30°C. In some embodiments, the conditions of (b) do not include contactingthe protein nanogels with the polymer at a temperature of greater than30° C.

In some embodiments, the conditions of (a) do not include contacting theprotein with the degradable linker in an organic solvent (e.g.,alcohol). In some embodiments, the conditions of (b) do not includecontacting the protein nanogels with the polymer in an organic solvent.

In some embodiments, the protein is a cytokine, growth factor, antibodyor antigen. For example, the protein may be a cytokine. In someembodiments, the cytokine is IL-2 or IL-2-Fc. In some embodiments, thecytokine is IL-15 or IL-15SA.

In some embodiments, the degradable linker is a redox responsive linker.In some embodiments, the redox responsive linker comprises a disulfidebond. In some embodiments, the degradable linker comprises or consistsof Formula I.

In some embodiments, the polymer is a hydrophilic polymer. Thehydrophilic polymer, in some embodiments, comprises polyethylene glycol(PEG). For example, the hydrophilic polymer may be a 4-arm PEG-NH₂polymer.

In some embodiments, the dry size of the carrier-free,biologically-active protein-polymer nanogels is less than 100 nm indiameter. For example, the dry size of the carrier-free,biologically-active protein-polymer nanogels may be 50-60 nm indiameter. In some embodiments, protein nanogels of a plurality, asprovided herein, are of similar dry size (e.g., within 1%, 2%, 3%, 4%,5% or 10% diameter of each other).

In some embodiments, the hydrodynamic size of the carrier-free,biologically-active protein-polymer nanogels is less than 100 nm indiameter. For example, the hydrodynamic size of the carrier-free,biologically-active protein-polymer nanogels may be 80-90 nm indiameter. In some embodiments, protein nanogels of a plurality, asprovided herein, are of similar hydrodynamic size (e.g., within 1%, 2%,3%, 4%, 5% or 10%, diameter of each other).

In some embodiments, the concentration of the protein in the aqueousbuffer is 10 mg/mL to 50 mg/mL (e.g., 10, 15, 20, 25, 30, 35, 40, 45 or50 mg/mL).

In some embodiments, the plurality of carrier-free, biologically-activeprotein-polymer nanogels is a monodispersed plurality of carrier-free,biologically-active protein-polymer nanogels.

In some embodiments, the carrier-free, biologically-activeprotein-polymer nanogels do not include albumin.

In some embodiments, the weight percentage of protein (e.g.,biologically-active protein, crosslinked protein) in the carrier-free,biologically-active protein-polymer nanogels is at least 75%. In someembodiments, the weight percentage of protein in the carrier-free,biologically-active protein-polymer nanogels is at least 80%. In someembodiments, the weight percentage of protein in the carrier-free,biologically-active protein-polymer nanogels is at least 85%. In someembodiments, the weight percentage of protein in the carrier-free,biologically-active protein-polymer nanogels is at least 90%.

Some aspects of the present disclosure provide methods of in vivoprotein delivery, comprising administering to a subject any one of thecompositions or nanogels provided herein.

In some embodiments, the subject has a disease. In some embodiments, thedisease is cancer, diabetes, an autoimmune disease or a cardiovasculardisease.

In some embodiments, the protein, under physiological conditions, isreleased in its native conformation from the nanogel and is biologicallyactive. In some embodiments, the specific activity of the releasedprotein is at least than 50% (e.g., at least 55%, 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100%) of the specific activityof the protein before it was crosslinked to another protein through adegradable linker.

Some aspects of the disclosure provide proteins reversibly linkedthrough a degradable linker to a polymerizable functional group. Suchproteins are considered herein to be reversibly modified proteins.

In some embodiments, the polymerizable functional group comprises silaneand/or a crosslinkable polymer. In some embodiments, the crosslinkablepolymer comprises poly(ethylene oxide), polylactic acid and/orpoly(lactic-co-glycolic acid). In some embodiments, the proteins arereversibly linked through a degradable linker to silane.

In some embodiments, proteins of the disclosure are cytokines, growthfactors, antibodies or antigens. In some embodiments, the cytokine isIL-2.

In some embodiments, the degradable linker comprises anN-hydroxysuccinimide ester. In some embodiments, the degradable linkeris a redox responsive linker. In some embodiments, the redox responsivelinker comprises a disulfide bond.

Other aspects of the disclosure provide pluralities of any reversiblymodified protein described herein.

In some embodiments, reversibly modified proteins in such pluralitiesare crosslinked.

Yet other aspects of the disclosure provide nanostructures that comprisea polymer and at least 50% w/w of a protein that is reversibly linkedthrough a degradable linker to a polymerizable functional group. “w/w”here means weight of protein to weight of nanostructure (e.g., nanogel).

In some embodiments, the polymerizable functional group comprises silaneand/or a crosslinkable polymer. In some embodiments, the crosslinkablepolymer comprises poly(ethylene oxide), polylactic acid and/orpoly(lactic-co-glycolic acid).

In some embodiments, the nanostructures comprise at least 75% w/w of aprotein that is reversibly linked to a polymerizable functional group.In some embodiments, the nanostructures comprise at least 80% w/w of aprotein that is reversibly linked to a polymerizable functional group.Also contemplated herein are nanostructures that comprise about 50% w/wto about 90% w/w of a protein that is reversibly linked to apolymerizable functional group. For example, in some embodiments, ananostructure may have about 50% w/w, about 55% w/w, about 60% w/w,about 65% w/w, about 70% w/w, about 75% w/w, about 80% w/w, about 85%w/w, or about 90% w/w of a protein that is reversibly linked to apolymerizable functional group.

In some embodiments, the protein is a cytokine, growth factor, antibodyor antigen. In some embodiments, the cytokine is IL-2.

In some embodiments, the nanostructures comprise a reactive group ontheir surface. In some embodiments, the reactive group is a maleimide,rhodamine or IR783 reactive group.

In some embodiments, the nanostructures are linked to a carrier cell. Insome embodiments, the carrier cell is a nucleated carrier cell. In someembodiments, the nucleated carrier cell is a T cell, a B cell, an NKcell or an NKT cell.

In some embodiments, the nanostructures are 20-500 nm in diameter. Insome embodiments, the nanostructures are 100-300 nm in diameter.

In some embodiments, the degradable linker comprises anN-hydroxysuccinimide ester. In some embodiments, the degradable linkeris a redox responsive linker. In some embodiments, the redox responsivelinker comprises a disulfide bond.

Still other aspects of the disclosure provide methods of producing ananostructure, the methods comprising modifying a protein with adegradable linker and polymerizable functional groups, and polymerizingthe polymerizable functional groups with a crosslinker and solublefluoride.

In some embodiments, the polymerizable functional group comprises silaneand/or a crosslinkable polymer. In some embodiments, the crosslinkablepolymer comprises poly(ethylene oxide), polylactic acid and/orpoly(lactic-co-glycolic acid).

In some embodiments, the soluble fluoride is sodium fluoride. In someembodiments, the soluble fluoride is potassium fluoride.

In some embodiments, the protein is a cytokine, growth factor, antibodyor antigen. In some embodiments, the cytokine is IL-2.

In some embodiments, the degradable linker comprises anN-hydroxysuccinimide ester. In some embodiments, the degradable linkeris a redox responsive linker. In some embodiments, the redox responsivelinker comprises a disulfide bond.

In some embodiments, the nanostructure is 20-500 nm in diameter. In someembodiments, the nanostructure is 100-300 nm in diameter.

In some embodiments, the methods further comprise modifying the surfaceof the nanostructure with a reactive group. In some embodiments, thereactive group is a maleimide, rhodamine or IR783 reactive group.

In some embodiments, the methods further comprise linking thenanostructure to a carrier cell. In some embodiments, the carrier cellis a nucleated carrier cell. In some embodiments, the nucleated carriercell is a T cell, a B cell, an NK cell or an NKT cell.

Further aspects of the disclosure provide methods of in vivo proteindelivery, comprising administering to a subject any of thenanostructures provided herein. In some embodiments, the methodscomprise administering to a subject a nanostructure that comprises aprotein reversibly linked through a degradable linker to silane.

In some embodiments, the subject has a condition or disease. In someembodiments, the condition or disease is cancer, diabetes, an autoimmunedisease, or a cardiovascular disease.

In some embodiments, the protein, under physiological conditions, isreleased in its native conformation from the nanostructure and isbiologically active.

The disclosure also provides a linker that comprises or consists ofFormula I:

The disclosure further provides reversibly modified protein conjugatesthat comprise Formula II:

Also provided herein are reversibly modified protein conjugates thatcomprise Formula III:

The linkers may be conjugated to the protein of interest at an aminegroup such as a terminal amine or an internal amine. Internal aminesinclude side chain amines such as lysine amines.

The disclosure further provides protein conjugates comprising FormulaIII:

wherein the protein is a cytokine such as, for example, IL-2.Unexpectedly, silica-based nanostructures with a high incorporationefficiency (e.g., >˜90%) and with high protein drug loading efficiency(e.g., >˜80%) are formed by the polymerization of proteins that arereversibly modified with silane. Thus, provided herein arenanostructures formed by the polymerization of protein conjugates ofFormula III with crosslinkers such as, for example, silane-PEG-silanepolymers.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows a schematic of a T lymphocyte engineering withsurface-conjugated interleukin-2 (IL-2)-loaded nanocapsules (NCs) fortargeted cancer therapy.

FIG. 2 shows a schematic of an example of synthesis and surfacefunctionalization of IL2-silica NCs.

FIGS. 3A-3D show an example of synthesis (FIG. 3A) and MALDI massspectrum (FIG. 3B) of IL2-fc-silane. Dynamic light scattering (DLS)(FIG. 3C) and scanning electron microscopy (SEM) (FIG. 3D) analysis ofthe IL2-silica NCs are also shown. IL-2-Fc is a bivalent fusion proteinin which the C terminus of murine wild-type IL-2 is linked to a mouseIgG2a Fc domain.

FIG. 4A shows incorporation efficiency and loading efficiency of IL2-fcin IL2-fc-silica NCs. Incorporation efficiency^([a])=conjugated IL2-fcin IL2-fc-NC/total IL2-fc added in reaction; Loading[^(b])=mass ofconjugated IL2-fc/total mass of IL2-fc-NC. FIG. 4B shows thatprotein-silica NC can release the incorporated protein in its originalform under physiological conditions. FIG. 4C shows release kinetics ofIL-2-fc from IL-2-fc-NC incubated in buffer of different pH at 37° C.for 48 h.

FIG. 5A shows a schematic of chemical conjugation of a maleimidefunctionalized IL-2-fc-silica NCs to an effector T cell surface via amaleimide-thiol coupling reaction. FIG. 5B shows a flow cytometryanalysis of T cells with surface-conjugated IL-2-silica NCs. FIGS. 5Cand 5D show an in vitro CD8+ T cell proliferation assay with free IL2-fcor IL2-fc-NC by manual counting and flow cytometry, respectively.

FIG. 6A shows a timeline of an in vivo CD8+ T cells expansion study.FIG. 6B shows images of mice with established lung metastases of B16F10melanoma received adoptive transfer of luciferase-expressing Pmel-1melanoma-specific CD8⁺ T-cells. T-cell expansion was followed over timeby bioluminescence imaging. FIG. 6C shows a flow cytometry analysis ofthe frequency of adoptively-transferred T-cells in the inguinal lymphnodes on day 6 after adoptive transfer.

FIG. 7 shows a schematic of various structures constructed withreversibly modified proteins.

FIGS. 8A-8B show schematics of the preparation of protein-PEG nanogels(NGs). FIG. 8A shows a 4 arm-PEG-NH2 that was reacted with Linker-1 toform the 4 arm-PEG-Linker-1, which bears NHS ester at the end of PEGpolymer chain. FIG. 8B shows a 4 arm-PEG-Linker-1 crosslinked by protein(e.g., IL-2), which has multiple amine groups forming IL-2-PEG nanogel.

FIG. 9A shows a schematic of one example of a method for preparing acovalently crosslinked protein nanogel. FIG. 9B shows a schematic of oneexample of a method for conjugating a protein nanogel to a cell surfaceand the release of intact, biologically-active protein.

FIGS. 10A-10C show an analysis of a covalently crosslinked proteinnanogel with HPLC equipped with a size exclusion column (FIG. 10A);transmission electron microscopy (FIG. 10B); and dynamic lightscattering (FIG. 10C) characterizations of the nanogels for size andmorphology.

FIG. 11A shows a schematic of a mechanism of the release of intact,biologically-active protein from a protein nanogel. FIG. 11B shows agraph of release kinetics of IL-2-Fc from a protein nanogel. FIG. 11Cshows glutathione (GSH) facilitated release of IL2-Fc, verified by HPLCequipped with a size exclusion column. FIG. 11D shows the releasedIL2-Fc and native IL2-Fc, analyzed with mass spectrum of Matrix-assistedlaser desorption/ionization.

FIGS. 12A-12B show the formation of other protein nanogels. Analyses ofthe human IL-15 superagonist (hIL-15Sa) nanogel (FIG. 12A) and nativemouse IL-2 (mIL-2) nongel (FIG. 12B) with HPLC equipped with a sizeexclusion column are shown.

FIG. 13 shows an image of vials containing bulk gel instead of nanogelswhen the protein concentration is too high (>50 mg/mL).

FIG. 14A shows a confocal microscope image of T cells withsurface-conjugated protein nanogels. FIG. 14B shows a flow cytometrygraph of controlled conjugation of IL-2-Fc nanogel to T cell surface atdifferent amounts.

FIGS. 15A-15C show in vivo CD8⁺ T cells expansion. FIG. 15A shows a timecourse of mice with established lung metastases of B16F10 melanoma thatwere lympho-depleted and that received adoptive transfer ofluciferase-expressing Pmel-1 melanoma-specific CD8⁺ T-cells with nofurther treatment, free IL2-Fc or surface-conjugated IL2-Fc nanogelrespectively in each group. FIG. 15B shows bioluminescence images ofT-cell expansion over time. FIG. 15C shows a graph quantifyingbioluminescence signal in the whole body of the mice.

FIG. 16 show the frequency of adoptively-transferred T cells andendogenous T cells in the inguinal lymph nodes (left) and blood (right)analyzed with flow cytometry 12 days (Day 12) after adoptive transfer.

FIGS. 17A-17D shows inhibition of metastatic tumors in lungs. FIG. 17Ashows representative images of harvested lungs from each group. FIG. 17Bshows a graph of the number of tumor nodules (counted manually) inlungs. FIG. 17C shows histological images of lung tissue sections thatwere graded for the severity of lung metastases. FIG. 17D shows a graphof the average grade of each group.

DETAILED DESCRIPTION OF THE INVENTION

Cancer immunotherapy, including adoptive T cell therapy, is a promisingstrategy to treat cancer because it harnesses a subject's own immunesystem to attack cancer cells. Nonetheless, a major limitation of thisapproach is the rapid decline in viability and function of thetransplanted T lymphocytes. In order to maintain high numbers of viabletumor-specific cytotoxic T lymphocytes in tumors, co-administration ofimmunostimulatory agents with transferred cells is necessary. When givensystemically at high doses, these agents could enhance the in vivoviability of transferred (i.e., donor) cells, improve the therapeuticfunction of transferred cells, and thus lead to overall improvedefficacy against cancer; however, high doses of such agents could alsoresult in life-threatening side effects. For example, the use ofinterleukin-2 (IL-2) as an adjuvant greatly supports adoptive T celltherapy of melanoma, where IL-2 provides key adjuvant signals totransferred T cells but also elicits severe dose-limiting inflammatorytoxicity and expands regulatory T cells (Tregs). One approach to focusadjuvant activity on the transferred cells is to genetically engineerthe transferred cells to secrete their own supporting factors. Thetechnical difficulty and challenges as well as the high cost forlarge-scale production of genetically engineered T lymphocytes havesignificantly limited the potential of this method in clinicalapplications, to date.

Provided herein, in some aspects, is a technology platform that permitssimple, safe and efficient delivery of biologically-active proteins(e.g., adjuvants such as IL-2) to therapeutic cells through chemicalconjugation of protein-loaded, carrier-free nanostructures orprotein-loaded silica-based nanostructures directly onto the plasmamembrane of transferred cells, enabling continuous pseudoautocrinestimulation of transferred cells in vivo. In some embodiments, proteinsof the disclosure are reversibly and covalently crosslinked to eachother through a degradable linker to form a nanostructure such that theintact, biologically-active proteins are released from the nanostructureunder physiological conditions, and optionally in the presence of areducing agent (e.g., glutathione). In other embodiments, proteins ofthe disclosure are reversibly modified and “ensheathed” intosilica-based nanostructures such that the intact, biologically-activeproteins are released from the nanostructure under physiologicalconditions, and optionally in the presence of a reducing agent (e.g.,glutathione). Surprisingly, nanostructures (e.g., carrier-free nanogelsand/or silica-based nanostructures) of the disclosure prevent proteasedegradation of the loaded protein and permit its sustained localrelease, thereby promoting the expansion of cytotoxic T cells andavoiding systemic toxicity associated with high-doses of some proteins(e.g., IL-2, IL-15). Unexpectedly, T cells with an optimal number ofnanostructures conjugated per cell maintain their cellular functions andcancer targeting and killing capability. Thus, the compositions andmethods of the disclosure can, in some embodiments, augment T cellexpansion and minimize systemic side effects of adjuvant drugs in vivo.

In addition to the foregoing, the present disclosure furthercontemplates other nanostructures that comprise other proteintherapeutics for purposes other than adjuvant effect onadoptively-transferred cells. Those of skill in the art will readilyrecognize that the disclosure has broader applications, as providedherein.

In some embodiments, proteins of protein nanostructures of the presentdisclosure are reversibly linked to each other through a degradablelinker (e.g., a disulfide linker) such that under physiologicalconditions, the linker degrades and releases the intact,biologically-active protein. In other embodiments, proteins ofnanostructures are reversibly linked to functional groups through adegradable linker such that under physiological conditions, the linkerdegrades and releases the intact, biologically-active protein. In eachinstance, the proteins are considered to be reversibly modified, asdescribed below.

A protein that is “reversibly linked to another protein” herein refersto a protein that is attached (e.g., covalently attached) to anotherprotein through a degradable linker. Such proteins are considered to belinked (e.g., crosslinked) to each other through the degradable linker.In some embodiments, nanostructures (e.g., nanogels) contain a single(e.g., single type of) biologically-active protein (e.g., IL-2, orIL-2-Fc), while in other embodiments, nanostructures contain more thanone (e.g., 2, 3, 4, 5 or more) of biologically-active protein (e.g., acombination of different proteins such as IL-2 and IL-15 (or IL-15SA)).For example, a protein nanogel may contain a combination of Protein Aand Protein B, wherein Protein A is linked to Protein A, Protein A islinked to Protein B and/or Protein B is linked to Protein B.

A protein that is “reversibly linked to a functional group,” or aprotein that is “reversibly modified,” herein refers to a protein thatis attached (e.g., covalently attached) to a functional group through adegradable linker. Such a protein may be referred to herein as a“protein conjugate” or a “reversibly modified protein conjugate”—theterms may be used interchangeably herein. It should be understood thatproteins and polymers each contain functional groups to which a proteincan be linked via a reversible linker (e.g., degradable linker such as adisulfide linker). Examples of protein conjugates and reversiblymodified proteins, as provided herein, include without limitation, aprotein reversibly linked (e.g., via a degradable linker) to anotherprotein, a protein reversibly linked to a polymer, and a proteinreversibly linked to another functional group. It should be understoodthat the term “protein” includes fusion proteins.

The degradable linkers provided herein, in some embodiments, comprise anN-hydroxysuccinimide ester, which is capable of reacting with proteinsat neutral pH (e.g., about 6 to about 8, or about 7) without denaturingthe protein. In some embodiments, the degradable linkers are “redoxresponsive” linkers, meaning that they degrade in the presence of areducing agent (e.g., glutathione, GSH) under physiological conditions(e.g., 20-40° C. and/or pH 6-8), thereby releasing intact protein fromthe compound to which it is reversibly linked. An example of adegradable linker for use in accordance with the present disclosure isthe following:

The linker of Formula I contains a disulfide, which is cleaved in thepresence of a reducing agent. For example, under physiologicalconditions, the disulfide bond of the linker of Formula I is cleaved byglutathione.

Proteins may be linked (e.g., covalently linked) to a degradable linkerthrough any terminal or internal —NH₂ functional group (e.g., side chainof a lysine). Thus, an intermediate species formed during the reversiblemodification of a protein with a degradable linker of Formula I is thefollowing:

Reversibly modified proteins provided herein can, in some embodiments,be formed or self-assemble into various nanostructures including,without limitation, protein-hydrophilic polymer conjugates (e.g.,reversibly modified with PEG; FIG. 7A), protein-hydrophobic polymerconjugates (e.g., reversibly modified PLA or PLGA; FIG. 7B), bulkcrosslinked protein hydrogels (FIG. 7C), crosslinked protein nanogelparticles (FIG. 7D), protein nanocapsules with different shell materials(e.g., silica; FIG. 7E), protein-conjugated nanoparticles (e.g.,liposome, micelle, polymeric nanoparticles, inorganic nanoparticles;FIG. 7F). Likewise, proteins crosslinked to each other, as providedherein, in some embodiments, can be formed or can self-assemble intoprotein nanostructures (e.g., FIG. 9A).

In some embodiments, protein nanostructures (e.g., protein nanogels,including protein-polymer nanogels) of the present disclosure do notcontain carrier proteins or other carrier molecules. For example, insome embodiments, protein nanostructures do not contain albumin (e.g.,bovine serum albumin (BSA)). Carrier proteins typically facilitate thediffusion and/or transport of different molecules. It should beunderstood that the term “carrier protein,” as used herein, refers to aprotein that does not adversely affect a biologically-active protein ofa protein nanostructure. In some embodiments, a carrier protein is aninert protein. Thus, in some embodiments, carrier proteins are notbiologically active. Nanostructures of the present disclosure, in someembodiments, do not require carrier proteins or other carrier moleculesto facilitate their transport to and into cells and tissue in vivo.

It should be understood that nanogels of the present disclosure, in someembodiments, contain one or more (e.g., 2, 3, 4, 5 or more) therapeuticproteins (e.g., IL-2 and/or IL-15 (or IL15-SA)) crosslinked to eachother through a degradable linker (e.g., disulfide linker). Suchnanogels no not contain an inert carrier protein, such as albumin.

Examples of proteins for use in accordance with the present disclosureinclude, without limitation, antibodies, single chain antibodies,antibody fragments, enzymes, co-factors, receptors, ligands,transcription factors and other regulatory factors, some antigens (asdiscussed below), cytokines, chemokines, and the like. These proteinsmay or may not be naturally occurring. Other proteins are contemplatedand may be used in accordance with the disclosure. Any of the proteinscan be reversibly modified through a redox responsive (e.g., disulfide)with a silane group to, for example, form a silica-based nanostructure.

In some embodiments, proteins of the disclosure are immunostimulatoryproteins. As used herein, an immunostimulatory protein is a protein thatstimulates an immune response (including enhancing a pre-existing immuneresponse) in a subject to whom it is administered, whether alone or incombination with another protein or agent. Examples of immunostimulatoryproteins that may be used in accordance with the disclosure include,without limitation, antigens, adjuvants (e.g., flagellin, muramyldipeptide), cytokines including interleukins (e.g., IL-2, IL-7, IL-15(or superagonist/mutant forms of these cytokines, such as, for example,IL-15SA), IL-12, IFN-gamma, IFN-alpha, GM-CSF, FLT3-ligand), andimmunostimulatory antibodies (e.g., anti-CTLA-4, anti-CD28, anti-CD3, orsingle chain/antibody fragments of these molecules). Otherimmunostimulatory proteins are contemplated and may be used inaccordance with the disclosure.

In some embodiments, proteins of the disclosure are antigens. Examplesof antigens that may be used in accordance with the disclosure include,without limitation, cancer antigens, self-antigens, microbial antigens,allergens and environmental antigens. Other protein antigens arecontemplated and may be used in accordance with the disclosure.

In some embodiments, proteins of the disclosure are cancer antigens. Acancer antigen is an antigen that is expressed preferentially by cancercells (i.e., it is expressed at higher levels in cancer cells than onnon-cancer cells) and, in some instances, it is expressed solely bycancer cells. Cancer antigens may be expressed within a cancer cell oron the surface of the cancer cell. Cancer antigens that may be used inaccordance with the disclosure include, without limitation,MART-1/Melan-A, gp100, adenosine deaminase-binding protein (ADAbp), FAP,cyclophilin b, colorectal associated antigen (CRC)-C017-1A/GA733,carcinoembryonic antigen (CEA), CAP-1, CAP-2, etv6, AML1, prostatespecific antigen (PSA), PSA-1, PSA-2, PSA-3, prostate-specific membraneantigen (PSMA), T cell receptor/CD3-zeta chain and CD20. The cancerantigen may be selected from the group consisting of MAGE-A1, MAGE-A2,MAGE-A3, MAGE-A4, MAGE-A5, MAGE-A6, MAGE-A7, MAGE-A8, MAGE-A9, MAGE-A10,MAGE-A11, MAGE-A12, MAGE-Xp2 (MAGE-B2), MAGE-Xp3 (MAGE-B3), MAGE-Xp4(MAGE-B4), MAGE-C1, MAGE-C2, MAGE-C3, MAGE-C4 and MAGE-C5. The cancerantigen may be selected from the group consisting of GAGE-1, GAGE-2,GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE-7, GAGE-8 and GAGE-9. The cancerantigen may be selected from the group consisting of BAGE, RAGE, LAGE-1,NAG, GnT-V, MUM-1, CDK4, tyrosinase, p53, MUC family, HER2/neu, p21ras,RCAS 1, α-fetoprotein, E-cadherin, α-catenin, β-catenin, γ-catenin,p120ctn, gp100Pmel117, PRAME, NY-ESO-1, cdc27, adenomatous polyposiscoli protein (APC), fodrin, Connexin 37, Ig-idiotype, p15, gp75, GM2ganglioside, GD2 ganglioside, human papilloma virus proteins, Smadfamily of tumor antigens, Imp-1, P1A, EBV-encoded nuclear antigen(EBNA)-1, brain glycogen phosphorylase, SSX-1, SSX-2 (HOM-MEL-40),SSX-1, SSX-4, SSX-5, SCP-1 and CT-7, CD20 and c-erbB-2. Other cancerantigens are contemplated and may be used in accordance with thedisclosure.

In some embodiments, proteins of the disclosure are antibodies orantibody fragments including, without limitation, bevacizumab(AVASTIN®), trastuzumab (HERCEPTIN®), alemtuzumab (CAMPATH®, indicatedfor B cell chronic lymphocytic leukemia), gemtuzumab (MYLOTARG®, hP67.6,anti-CD33, indicated for leukemia such as acute myeloid leukemia),rituximab (RITUXAN®), tositumomab (BEXXAR®, anti-CD20, indicated for Bcell malignancy), MDX-210 (bispecific antibody that binds simultaneouslyto HER-2/neu oncogene protein product and type I Fc receptors forimmunoglobulin G (IgG) (Fc gamma RI)), oregovomab (OVAREX®, indicatedfor ovarian cancer), edrecolomab (PANOREX®), daclizumab (ZENAPAX®),palivizumab (SYNAGIS®, indicated for respiratory conditions such as RSVinfection), ibritumomab tiuxetan (ZEVALIN®, indicated for Non-Hodgkin'slymphoma), cetuximab (ERBITUX®), MDX-447, MDX-22, MDX-220 (anti-TAG-72),IOR-C5, IOR-T6 (anti-CD1), IOR EGF/R3, celogovab (ONCOSCINT® OV103),epratuzumab (LYMPHOCIDE®), pemtumomab (THERAGYN®) and Gliomab-H(indicated for brain cancer, melanoma). Other antibodies and antibodyfragments are contemplated and may be used in accordance with thedisclosure.

Proteins of the disclosure may be modified in a binary solvent that iscompatible with proteins. For example, in some embodiments, a binarysolvent includes aqueous buffer and a water-miscible organic solvent,such as phosphate buffered saline (PBS) and dimethyl sulfoxide (DMSO),and is used for reversibly modifying a protein with a degradable linker.The ratio of the aqueous buffer (e.g., PBS) to organic phase (e.g.,DMSO) may be within a range of about 50:1 to about 20:1. In someembodiments, the ratio of inorganic phase to organic phase is about 30:1to about 20:1, or about 25:1 (e.g., 500 μL:20 μL). In some embodiments,the organic solvent is less than 5% of the total volume of the binarybuffer or the reaction containing the binary buffer.

A “polymerizable functional group,” as used herein, refers to a group ofatoms and bonds that can chemically react to form a polymer chain ornetwork. A “polymer” refers to a chain or network of repeating units ora mixture of different repeating units. As used herein, a polymer isitself a functional group. Examples of polymerizable functional groupsfor use in accordance with the disclosure include, without limitation,silane, ethylene oxide, lactic acid, lactide, glycolic acid,N-(2-hydroxypropyl)methacrylamide, silica, poly(ethylene oxide),polylactic acid, poly(lactic-co-glycolic acid), polyglutamate,polylysine, cyclodextrin and dextran chitosan. Other polymerizablefunctional groups are contemplated and may be used in accordance withthe disclosure. It should be understood, however, that a “polymer,” asused herein, is not a protein (is a non-protein), peptide (is anon-peptide) or amino acid (is a non-amino acid).

It should be understood that the term “polymer” encompasses“co-polymer.” That is, a polymer may comprise a mixture of differentfunctional groups (e.g., silane-PEG-silane), including shorter polymersor co-polymers. The functional groups are typically polymerized underprotein-compatible, neutral conditions. Thus, in some embodiments,polymerization of the functional groups occurs in an at least partiallyaqueous solution at about pH 6 to about pH 8. For example,polymerization of the functional groups can occur at pH 6, pH 6.5, pH 7,pH 7.5 or pH 8. In some embodiments, polymerization of the functionalgroups occurs at about pH 7.

In some embodiments, the polymerization reaction is catalyzed by sodiumfluoride, potassium fluoride or any other soluble fluoride.

Exemplary polymers that can be reversibly linked to proteins and/or usedto form nanostructures (e.g., nanocapsules, nanogels, hydrogels)include, without limitation, aliphatic polyesters, poly (lactic acid)(PLA), poly (glycolic acid) (PGA), co-polymers of lactic acid andglycolic acid (PLGA), polycarprolactone (PCL), polyanhydrides,poly(ortho)esters, polyurethanes, poly(butyric acid), poly(valericacid), and poly(lactide-co-caprolactone), and natural polymers such asalginate and other polysaccharides including dextran and cellulose,collagen, chemical derivatives thereof, including substitutions,additions of chemical groups such as for example alkyl, alkylene,hydroxylations, oxidations, and other modifications routinely made bythose skilled in the art), albumin and other hydrophilic proteins, zeinand other prolamines and hydrophobic proteins, copolymers and mixturesthereof. In general, these materials degrade either by enzymatichydrolysis or exposure to water in vivo, by surface or bulk erosion.Other polymers are contemplated and may be used in accordance with thedisclosure.

In some aspects of the disclosure, proteins are reversibly linked tohydrophilic polymers such as, for example, polyethylene glycol (PEG)(FIG. 7A and FIGS. 9A-9B).

In other aspects of the disclosure, proteins are reversibly linked tohydrophobic polymers such as, for example, polylactic acid (PLA) and/orpoly(lactic-co-glycolic acid) (PLGA). These protein-hydrophobic polymerconjugates can, in some embodiments, self-assemble into nanoparticles(FIGS. 7B and 7F).

The protein conjugates of the present disclosure, in some embodiments,may be crosslinked to form a hydrogel network (FIG. 7C), nanogelparticle (FIG. 7D), or protein nanogel (FIG. 9A), all of which areherein considered to be “nanostructures.”

A protein “nanostructure,” as used herein, refers to a plurality ofcrosslinked protein conjugates (e.g., protein reversibly linked througha degradable linker to a functional group or polymer, or “reversiblymodified”) wrapped in a polymer-based, or silica, nanoshell (FIG. 7E).The nanoshell is formed, in some embodiments, by polymerizing functionalgroups (e.g., silanes) of a protein conjugate with a crosslinker (e.g.,silane-PEG-silane) in the presence of a catalyst (e.g., NaF). An exampleof a protein nanostructure is a “protein nanogel,” which refers to aplurality of proteins crosslinked (e.g., reversibly and covalentlycrosslinked) to each other through a degradable linker (see, e.g., FIG.9A). In some embodiments, proteins of a nanogel are crosslinked (e.g.,reversibly and covalently crosslinked) to a polymer (e.g., a hydrophilicpolymer such as polyethylene glycol (PEG); see, e.g., FIG. 9A). Thepolymer, in some embodiments, may be crosslinked to the surface of thenanogel (e.g., to proteins exposed at the surface of the nanogel).

The size of a protein nanogel may be determined at least two ways: basedon its “dry size” and based on its “hydrodynamic size.” The “dry size”of a protein nanogel refers to the diameter of the nanogel as a drysolid. The “hydrodynamic size” of a protein nanogel refers to thediameter of the nanogel as a hydrated gel (e.g., a nanogel in an aqueousbuffer). The dry size of a nanogel may be determined, for example, bytransmission electron microscopy, while the hydrodynamic size of thenanogel may be determined, for example, by dynamic light scattering.

In some embodiments, the dry size of a nanogel is less than 100 nm. Insome embodiments, the dry size of a nanogel is less than 95 nm, lessthan 90 nm, less than 85 nm, less than 80 nm, less than 75 nm, less than70 nm, less than 65 nm, or less than 60 nm. In some embodiments, the drysize of a nanogel is 40 to 90 nm, 40 to 80 nm, 40 to 70 nm, 40 to 60 nm,50 to 90 nm, 60 to 80 nm, 50 to 70 nm, or 50 to 60 nm. In someembodiments, the dry size of a nanogel is 40 nm, 45 nm, 50 nm, 55 nm, 60nm, 65 nm, 70 nm, 75 nm, 80 nm, 85 nm, 90 nm or 95 nm.

In some embodiments, the average dry size of a nanostructure (e.g.,nanogel) within a plurality of nanostructures is less than 100 nm. Insome embodiments, the average dry size of a nanostructure within such aplurality varies by no more than 5% or 10%. In some embodiments, theaverage dry size of a nanostructure (e.g., nanogel) within a pluralityof nanostructures is less than 95 nm, less than 90 nm, less than 85 nm,less than 80 nm, less than 75 nm, less than 70 nm, less than 65 nm, orless than 60 nm. In some embodiments, the average dry size of ananostructure (e.g., nanogel) within a plurality of nanostructures is 40to 90 nm, 40 to 80 nm, 40 to 70 nm, 40 to 60 nm, 50 to 90 nm, 60 to 80nm, 50 to 70 nm, or 50 to 60 nm. In some embodiments, the dry size of ananogel is 40 nm, 45 nm, 50 nm, 55 nm, 60 nm, 65 nm, 70 nm, 75 nm, 80nm, 85 nm, 90 nm or 95 nm.

In some embodiments, the hydrodynamic size of a nanogel is less than 100nm. In some embodiments, the dry size of a nanogel is less than 95 nm,less than 90 nm, less than 80 nm, less than 85 nm, or less than 75 nm.In some embodiments, the hydrodynamic size of a nanogel is 70 to 90 nm,70 to 85 nm, 70 to 80 nm, 75 to 90 nm, 75 to 85 nm, 75 to 80 nm, 80 to90 nm, 80 to 85 nm or 85 to 90 nm. In some embodiments, the hydrodynamicsize of a nanogel is 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, or 95 nm. Insome embodiments, the hydrodynamic size of a nanogel is 80 nm, 81 nm, 82nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm or 90 nm.

In some embodiments, the average hydrodynamic size of a nanostructure(e.g., nanogel) within a plurality of nanostructures is less than 100nm. In some embodiments, the average hydrodynamic size of ananostructure within such a plurality varies by no more than 5% or 10%.In some embodiments, the average hydrodynamic size of a nanostructure(e.g., nanogel) within a plurality of nanostructures is less than 95 nm,less than 90 nm, less than 80 nm, less than 85 nm, or less than 75 nm.In some embodiments, the average hydrodynamic size of a nanostructure(e.g., nanogel) within a plurality of nanostructures is 70 to 90 nm, 70to 85 nm, 70 to 80 nm, 75 to 90 nm, 75 to 85 nm, 75 to 80 nm, 80 to 90nm, 80 to 85 nm or 85 to 90 nm. In some embodiments, the averagehydrodynamic size of a nanostructure (e.g., nanogel) within a pluralityof nanostructures is 70 nm, 75 nm, 80 nm, 85 nm, 90 nm, or 95 nm. Insome embodiments, the average hydrodynamic size of a nanostructure(e.g., nanogel) within a plurality of nanostructures is 80 nm, 81 nm, 82nm, 83 nm, 84 nm, 85 nm, 86 nm, 87 nm, 88 nm, 89 nm or 90 nm.

In some embodiments, nanostructures are provided in a dry, solid form,such as a lyophilized form. In other embodiments, nanostructures areprovided in a hydrated form, such as in aqueous or otherwise liquidsolution.

Nanostructures, in some embodiments, are substantially sphericalnanocapsules or nanoparticles. In some embodiments, the diameter of ananostructure ranges from 1-1000 nanometers (nm). In some embodiments,the diameter ranges in size from 20-750 nm, or from 20-500 nm, or from20-250 nm. In some embodiments, the diameter ranges in size from 50-750nm, or from 50-500 nm, or from 50-250 nm, or from about 100-300 nm. Insome embodiments, the diameter is about 100, about 150, about 200 nm,about 250 nm, or about 300 nm.

As discussed herein, the nanostructures may be modified or synthesizedto comprise one or more reactive groups on their exterior surface forreaction with reactive groups on cell carriers (e.g., T cells). Thesenanostructure reactive groups include, without limitation,thiol-reactive maleimide head groups, haloacetyl (e.g., iodoacetyl)groups, imidoester groups, N-hydroxysuccinimide esters, pyridyldisulfide groups, and the like. These reactive groups react with groupson the carrier cell surface and, thus, the nanostructures are bound tothe cell surface. It will be understood that when surface modified inthis manner, the nanostructures are intended for use with specificcarrier cells having “complementary” reactive groups (i.e., reactivegroups that react with those of the nanostructures). In someembodiments, the nanostructures will not integrate into the lipidbilayer that comprises the cell surface. Typically, the nanostructureswill not be phagocytosed (or internalized) by the carrier cells.

In some embodiments the nanostructures do not comprise antibodies orantibody fragments on their surface, while in other embodiments they do.In some embodiments the nanostructures do not comprise antibodies orantibody fragments that are specific to T cell surface moieties (orexogenous moieties coated onto a T cell surface such other antibodies orantibody fragments), while in other embodiments they do. Thus, in someembodiments the nanostructures themselves do not stimulate carrier cellactivation simply by binding to the carrier cell. In other embodiments,however, the nanostructures do stimulate carrier cell activation bybinding to the carrier cell (e.g., binding of the nanostructures resultsin crosslinking of cell surface moieties and this activates the carriercell).

The nanostructures may be covalently conjugated (or attached or bound,as the terms are used interchangeably herein), or they may benon-covalently conjugated to the carrier cells. Covalent conjugationtypically provides a more stable (and thus longer) association betweenthe nanostructures and the carrier cells. Covalent conjugation, in someembodiments, also can provide stability and thus more sustainedlocalized delivery of agents in vivo. Non-covalent conjugation includes,without limitation, absorption onto the cell surface and/or lipidbilayer of the cell membrane.

In some instances, covalent attachment can be achieved in a two-stepprocess in which carrier cells are first incubated withmaleimide-bearing nanostructures to allow conjugation to the cellsurface, followed by in situ PEGylation with thiol-terminatedpoly(ethylene glycol) (PEG) to cap remaining maleimide groups of theparticles and avoid particle-mediated crosslinking of cells.

Carrier Cells

The carrier cells are the cells to which the nanostructures areconjugated and which, when administered in vivo, preferably home totarget site(s). Suitable target cells are chosen based on their homingpotential, their cell surface phenotype (for conjugation to thenanoparticles), and their ability to carry but not significantlyendocytose the nanostructures. In some embodiments described herein, Tcells are suitable carrier cells. The T cells may be CD4+ or CD8+ Tcells. Other suitable cells include B cells, NK cells, NK T cells, andhematopoietic progenitor cells including, without limitation, murinelineage-negative, Sca-1-positive and c-kit-positive cells and theirhuman counterparts. Substantial levels of free thiol (—SH) groups existon the surfaces of T cells, B cells and hematopoietic progenitor cells(data not shown), thereby facilitating conjugation of nanocapsules tosuch cells.

Carrier cells, in some embodiments, can extravasate from blood vessels(particularly when administered by intravenous injection) and therebyenter target tissues or organs. Red blood cells typically are not ableto exit the blood stream. Accordingly, one important class of carriercells includes nucleated carrier cells. Thus, in some embodiments,carrier cells are not red blood cells. In other embodiments, carriercells are red blood cells.

Some embodiments of the present disclosure refer to isolated carriercells. Isolated carrier cells are cells that have been separated fromthe environment in which they naturally occur (i.e., they are notpresent in vivo). T cells in vitro are an example of an isolated cell.It should be understood that carrier cells may be isolated from their invivo environment, conjugated to nanostructures of the presentdisclosure, and then re-introduced in vivo. Such carrier cells are stillconsidered to be isolated cells.

The carrier cells, in some embodiments, are autologous to a subjectbeing treated. In other embodiments, the carrier cells arenon-autologous (yet preferably MHC matched cells).

The carrier cells typically have a half-life in vivo, followingadministration (or re-infusion, in some instances) of at least 48 hours,at least 3 days, at least 4 days, at least 5 days, at least 6 days, atleast 7 days, or more.

The carrier cells, in some embodiments, are genetically engineered toexpress one or more factors including, without limitation,co-stimulatory molecules or receptors including chimeric receptors. Inother embodiments, the carrier cells are not genetically engineered. Insome embodiments, the carrier cells are isolated and naturally occurring(i.e., they have not been genetically or otherwise engineered).

Depending on their nature and function, the carrier cells, in someembodiments, are manipulated prior to conjugation with thenanostructures. The carrier cells, however, need not be surface-modifiedin order to facilitate conjugation of the nanostructures. In some ofembodiments, instead, reactive groups that normally exist on the carriercell surface are used without having to incorporate reactive groups orother entities onto the cell surface. As a result, such carrier cells donot require the presence of exogenous entities such as antibodies orantibody fragments, among others, on their surface in order to conjugateto nanostructures.

Such manipulation may also involve activation of the carrier cells, asis routinely performed for T cells. The carrier cells may, in someembodiments, be expanded and/or activated (or stimulated, as the termsare used interchangeably herein) in vitro prior to mixing withnanostructures. Expansion and activation protocols will vary dependingon the carrier cell type but can include incubation with one or morecytokines, incubation with one or more cell types, and incubation withone or more antigens. If the carrier cell is a T cell, then activationmay be performed by incubating the T cells with IL-2, IL-15, IL-15superagonist, costimulatory molecules such as B7, B7.2, CD40, antibodiesto various T cell surface molecules including antibodies to cell surfacereceptors, anti-CD3 antibodies, anti-CD28 antibodies, anti-CTLA-4antibodies, anti-CD40L antibodies, and the like. In some embodiments,the carrier cells and more particularly the T cells, are not coated withexogenous antibodies on their cell surface (i.e., the cells have notbeen contacted with antibodies or antibody fragments in vitro prior toadministration).

Expansion may be measured by proliferation assays involvingincorporation of radiolabeled nucleotides such as tritiated thymidine.Activation may be measured by production of cytokines such as IL-2,gamma-IFN, IL-1, IL-4, IL-6 and TNF, among others. Other ways ofmeasuring expansion and activation are known in the art and may be usedin accordance with the disclosure.

Carrier cells may be selected prior to administration to a subject inorder to enrich and thus administer higher numbers of such cells insmaller volumes and/or to remove other, potentially unwanted, cells fromthe administered composition. Selection may involve positive or negativeselection including, for example, column or plate based enrichmentprotocols that are known in the art.

T and B cells may be harvested from the peripheral blood of a subject.

Hematopoietic progenitor cells may be obtained from a number of sourcesincluding but not limited to cord blood, bone marrow, mobilizedperipheral blood and, in some instances, differentiated embryonic stemcells.

Hematopoietic progenitor cells have been characterized in the art. Suchcells in the human generally have minimally a CD34+ phenotype, althoughthey may also be CD59⁺, Thy1/CD90⁺, CD38^(lo/neg), CD33⁻, and/orc-kit/CD117⁺. They also are characterized as not expressing lineagespecific markers. They can be harvested from bone marrow, cord blood orperipheral blood using affinity columns, magnetic beads, fluorescenceactivated cell sorting (FACS), some combination thereof, and the like.These cells have the ability to repopulate one or more hematopoieticlineages upon transplantation. Preferably, these cells repopulate morethan one lineage, and even more preferably, all lineages. Repopulationor population of lineages as used herein refers to the differentiationof the stem cell into one or more lineages such that progeny of the stemcell contribute to the make-up of that lineage in the subject. It doesnot, however, require that the entire lineage compartment derive fromthe transplanted cells, however in some instances this may occur.

Isolated stem cells may be obtained by fractionating a heterogeneouscell population according to one or more markers, including by notlimited to cell surface markers.

The carrier cells may be eukaryotic cells, such as mammalian cells(e.g., human cells). Alternatively, they may be non-mammalian cells. Instill other embodiments, the carrier cells may be prokaryotic cells(e.g., bacterial cells). Several bacterial cell types are of particularinterest. For example, attenuated salmonella typhimurium is under studyas a candidate vector for oral vaccine delivery (Xiang et al., ImmunolRev 222:117, 2008; and Iweala et al., J Immunol 183(4):2252, 2009) andengineered E. coli bacteria have been shown to be capable of specifichoming to poorly oxygenated tumors (Cheong et al., Science314(5803):1308, 2006). Bacteria offer new modes of administration andtissue site targeting possibilities, such as oral administration and theability to target therapeutics to the gut and gut-associated lymphoidtissues. Such microbial vectors may offer advantages relative toautologous host cells in terms of creating off-the-shelf ready-to-usecell-nanoparticles systems. Particles conjugation to microbes can beachieved using the same suite of chemical strategies described formammalian cells. In some instances, temporary removal of flagellar coatsof microbes (e.g., via simple mechanical shearing as described by Rosuet al., J Bacteriol 188(14):5196, 2006) can be used to achieve optimalconjugation of particles to microbe cell bodies.

Methods

Provided herein are methods of producing nanostructures. An example of ananostructure is a protein nanogel, such as a protein nanogel thatcontains intact, biologically-active proteins but does not contain acarrier (e.g., albumin, BSA). In some embodiments, a method of producinga carrier-free, biologically-active protein nanogel comprises contactinga protein with a degradable linker under conditions that permitreversible covalent crosslinking of proteins to each other through thedegradable linker, thereby producing a carrier-free, biologically-activeprotein nanogel. In some embodiments, a method further comprisescontacting the protein nanogel with a polymer under conditions thatpermit crosslinking of the polymer to proteins of the protein nanogel,thereby producing a carrier-free, biologically-active protein-polymernanogel. In some embodiments, a plurality of protein nanogels or aplurality of protein-polymer nanogels is produced.

Typically, conditions that permit reversible covalent crosslinking ofproteins to each other through a degradable linker include contactingthe proteins with degradable linkers at a temperature of 4° C. to 25° C.(e.g., 4° C., 5° C., 6° C., 7° C., 8° C., 9° C., 10° C., 11° C., 12° C.,13° C., 14° C., 15° C., 16° C., 17° C., 18° C., 19° C., 20° C., 21° C.,22° C., 23° C., 24° C. or 25° C.). In some embodiments, proteins areincubated with the degradable linkers in an aqueous buffer (e.g., PBS)at a temperature of 4° C. to 25° C. (e.g., room temperature). In someembodiments, proteins are incubated with the degradable linkers in anaqueous buffer (e.g., PBS) at a temperature of no greater than 30° C. Insome embodiments, conditions that permit reversible covalentcrosslinking of proteins to each other through a degradable linkerinclude contacting proteins with degradable linkers for 30 minutes totwo hours, or 30 minutes to one hour (e.g., 30, 35, 40, 45, 50, 55 or 60minutes). In some embodiments, proteins are incubated with thedegradable linkers in an aqueous buffer (e.g., PBS) for 30 minutes totwo hours, or 30 minutes one hour.

In some embodiments, the concentration of the protein in the aqueousbuffer is 10 mg/mL to 50 mg/mL. For example, the concentration of theprotein in an aqueous buffer may be 10 mg/mL, 15 mg/mL, 20 mg/mL, 25mg/mL, 30 mg/mL, 35 mg/mL, 40 mg/mL, 45 mg/mL or 50 mg/mLprotein/aqueous buffer).

In some embodiments, the weight percentage of protein in a carrier-free,biologically-active protein nanogel or protein-polymer nanogel is atleast 75% w/w. For example, the weight percentage of protein in thecarrier-free, biologically-active protein-polymer nanogels is at least80% w/w, at least 85% w/w, at least 90% w/w, or at least 95% w/w. Insome embodiments, the weight percentage of protein in a carrier-free,biologically-active protein nanogel or protein-polymer nanogel is 75%w/w to 90% w/w, 80% w/w to 90% w/w, or 85% w/w to 90% w/w.

Conditions that permit crosslinking of a polymer to proteins of aprotein nanogel include contacting the protein nanogel with a polymer ata temperature of 4° C. to 25° C. (e.g., 4° C., 5° C., 6° C., 7° C., 8°C., 9° C., 10° C., 11° C., 12° C., 13° C., 14° C., 15° C., 16° C., 17°C., 18° C., 19° C., 20° C., 21° C., 22° C., 23° C., 24° C. or 25° C.).In some embodiments, protein nanogels are incubated with the polymers inan aqueous buffer (e.g., PBS) at a temperature of 4° C. to 25° C. (e.g.,room temperature). In some embodiments, protein nanogels are incubatedwith the polymers in an aqueous buffer (e.g., PBS) at a temperature ofno greater than 30° C. In some embodiments, conditions that permitcrosslinking of a polymer to proteins of a protein nanogel includecontacting the protein nanogel with a polymer for 30 minutes to twohours, or 30 minutes to one hour (e.g., 30, 35, 40, 45, 50, 55 or 60minutes). In some embodiments, protein nanogels are incubated with thepolymer in an aqueous buffer (e.g., PBS) for 30 minutes to two hours, or30 minutes one hour.

In some embodiments, methods of the present disclosure specificallyexclude contacting a protein with a degradable linker in the presence ofan organic solvent (e.g., an alcohol such as ethanol or isopropanol). Insome embodiments, methods of the present disclosure specifically excludecontacting a protein nanogel with a polymer in the presence of anorganic solvent (e.g., an alcohol such as ethanol or isopropanol).Organic solvents may adversely affect the biological activity of theproteins.

Other methods of producing nanostructures of the present disclosure maycomprise modifying a protein with a degradable linker and polymerizablefunctional groups, and polymerizing the polymerizable functional groupswith a crosslinker and soluble fluoride.

Proteins of the disclosure may be modified with, or conjugated to, adegradable linker such as, for example, a redox responsive linker. Themodification may, in some embodiments, be a covalent modification. FIG.3A illustrates one example of a protein modification scheme. In thisexample, a protein is covalently conjugated, through a degradablelinker, to silane.

Polymerizable functional groups may be polymerized with a crosslinker inthe presence of a soluble fluoride catalyst. In some embodiments, thecrosslinker is a polymer (e.g., silane-PEG-silane). In some embodiments,the soluble fluoride is sodium fluoride. In some embodiments, thesoluble fluoride is potassium fluoride.

The disclosure also provides methods of administering protein conjugatesand nanostructures in vivo to subjects.

The methods of the disclosure can be practiced in virtually any subjecttype that is likely to benefit from delivery of proteins as contemplatedherein. Human subjects are preferred subjects in some embodiments.Subjects also include animals such as household pets (e.g., dogs, cats,rabbits, ferrets), livestock or farm animals (e.g., cows, pigs, sheep,chickens and other poultry), horses such as thoroughbred horses,laboratory animals (e.g., mice, rats, rabbits), and the like. Subjectsalso include fish and other aquatic species.

The subjects to whom protein conjugates are delivered may be normal, orhealthy, subjects. Alternatively they may have or may be at risk ofdeveloping a condition that can be diagnosed or that can benefit fromdelivery of one or more particular proteins.

Such conditions include cancer (e.g., solid tumor cancers), autoimmunedisorders, allergies or allergic conditions, asthma, transplantrejection, and the like.

Tests for diagnosing various conditions embraced by the presentdisclosure are known in the art and will be familiar to the ordinarymedical practitioner. These laboratory tests include without limitationmicroscopic analyses, cultivation dependent tests (such as cultures),and nucleic acid detection tests. These include wet mounts,stain-enhanced microscopy, immune microscopy (e.g., FISH), hybridizationmicroscopy, particle agglutination, enzyme-linked immunosorbent assays,urine screening tests, DNA probe hybridization, serologic tests, etc.The medical practitioner will generally also take a full history andconduct a complete physical examination in addition to running thelaboratory tests listed above.

A subject having a cancer is a subject who has detectable cancer cells.A subject at risk of developing a cancer is a subject who has a higherthan normal probability of developing cancer. These subjects include,for instance, subjects having a genetic abnormality that has beendemonstrated to be associated with a higher likelihood of developing acancer, subjects having a familial disposition to cancer, subjectsexposed to cancer causing agents (e.g., carcinogens) such as tobacco,asbestos, or other chemical toxins, and subjects previously treated forcancer and in apparent remission.

Cancer

The present disclosure contemplates administration of reversiblymodified protein conjugates and/or protein nanostructures to subjectshaving or at risk of developing a cancer including, for example, a solidtumor cancer. The cancer may be carcinoma, sarcoma or melanoma.Carcinomas include, without limitation, to basal cell carcinoma, biliarytract cancer, bladder cancer, breast cancer, cervical cancer,choriocarcinoma, CNS cancer, colon and rectum cancer, kidney or renalcell cancer, larynx cancer, liver cancer, small cell lung cancer,non-small cell lung cancer (NSCLC, including adenocarcinoma, giant (oroat) cell carcinoma, and squamous cell carcinoma), oral cavity cancer,ovarian cancer, pancreatic cancer, prostate cancer, skin cancer(including basal cell cancer and squamous cell cancer), stomach cancer,testicular cancer, thyroid cancer, uterine cancer, rectal cancer, cancerof the respiratory system, and cancer of the urinary system. Othercancers are known and are contemplated herein.

Sarcomas are rare mesenchymal neoplasms that arise in bone(osteosarcomas) and soft tissues (fibrosarcomas). Sarcomas includewithout limitation liposarcomas (including myxoid liposarcomas andpleiomorphic liposarcomas), leiomyosarcomas, rhabdomyosarcomas,malignant peripheral nerve sheath tumors (also called malignantschwannomas, neurofibrosarcomas, or neurogenic sarcomas), Ewing's tumors(including Ewing's sarcoma of bone, extraskeletal (i.e., not bone)Ewing's sarcoma, and primitive neuroectodermal tumor), synovial sarcoma,angiosarcomas, hemangiosarcomas, lymphangiosarcomas, Kaposi's sarcoma,hemangioendothelioma, desmoid tumor (also called aggressivefibromatosis), dermatofibrosarcoma protuberans (DFSP), malignant fibroushistiocytoma (MFH), hemangiopericytoma, malignant mesenchymoma, alveolarsoft-part sarcoma, epithelioid sarcoma, clear cell sarcoma, desmoplasticsmall cell tumor, gastrointestinal stromal tumor (GIST) (also known asGI stromal sarcoma), and chondrosarcoma.

Melanomas are tumors arising from the melanocytic system of the skin andother organs. Examples of melanoma include without limitation lentigomaligna melanoma, superficial spreading melanoma, nodular melanoma, andacral lentiginous melanoma.

The cancer may be a solid tumor lymphoma. Examples include Hodgkin'slymphoma, Non-Hodgkin's lymphoma, and B cell lymphoma.

The cancer may be, without limitation, bone cancer, brain cancer, breastcancer, colorectal cancer, connective tissue cancer, cancer of thedigestive system, endometrial cancer, esophageal cancer, eye cancer,cancer of the head and neck, gastric cancer, intra-epithelial neoplasm,melanoma neuroblastoma, Non-Hodgkin's lymphoma, non-small cell lungcancer, prostate cancer, retinoblastoma or rhabdomyosarcoma.

Compositions

Compositions, including pharmaceutical compositions, comprising proteinnanostructures (e.g., protein nanogels) are provided herein. Acomposition can be administered to a subject inpharmaceutically-acceptable amounts and in pharmaceutically-acceptablecompositions. The term “pharmaceutically acceptable” means a non-toxicmaterial that does not interfere with the effectiveness of thebiological activity of the active ingredients (e.g., biologically-activeproteins of the nanostructures). Such compositions may, in someembodiments, contain salts, buffering agents, preservatives, andoptionally other therapeutic agents.

Pharmaceutical compositions also may contain, in some embodiments,suitable preservatives.

Pharmaceutical compositions may, in some embodiments, be presented inunit dosage form and may be prepared by any of the methods well-known inthe art of pharmacy.

Pharmaceutical compositions suitable for parenteral administration, insome embodiments, comprise a sterile aqueous or non-aqueous preparationof the nanostructures, which is, in some embodiments, isotonic with theblood of the recipient subject. This preparation may be formulatedaccording to known methods. A sterile injectable preparation also may bea sterile injectable solution or suspension in a non-toxicparenterally-acceptable diluent or solvent.

Pharmaceutical compositions of the present disclosure are administered,in some embodiments, by a conventional route, including injection or bygradual infusion over time. Administration may, for example, be oral,intravenous, intraperitoneal, intramuscular, intracavity, intratumor, ortransdermal.

Pharmaceutical compositions of the present disclosure are administered,in some embodiments, in effective amounts. An “effective amount” is thatamount of any of the nanostructure provided herein that alone, ortogether with further doses and/or other therapeutic agents, produces adesired response (e.g., pseudoautocrine stimulation, augment T cellexpansion and minimize systemic side effects of adjuvant drugs in vivo).

Pharmaceutical compositions of the present disclosure, in someembodiments, may be sterile and contain an effective amount of ananostructure (e.g., nanogel), alone or in combination with anotheragent, for producing the desired response in a unit of weight or volumesuitable for administration to a subject (e.g., human subject). Theresponse can, for example, be measured by determining the physiologicaleffects of the nanostructure composition.

The doses of compositions administered to a subject may be chosen inaccordance with different parameters, in particular in accordance withthe mode of administration used and the state of the subject. Otherfactors include the desired period of treatment. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that subject/patienttolerance permits.

EXAMPLES Example 1

In the context of adoptive T cell therapy for cancer treatment, adjuvantcytokine drug, IL 2, provides key adjuvant signals to donor T cells butalso elicits severe dose-limiting inflammatory toxicity and expandsregulatory T cells (T_(reg)s). Provided herein is a delivery method tosafely and efficiently target IL-2 to therapeutic cells with minimaltoxicity.

FIG. 1 illustrates an example of a method of preparing protein-silicananocapsules (NCs). Polymerizable silane groups were first conjugated toIL-2 through a redox responsive linker (Formula I) to prepareIL-2-silane (FIG. 2, I). The modified IL-2 was further reacted with(3-Aminopropyl)triethoxysilane to functionalize IL-2 with polymerizablesilane group on the protein surface. Subsequent polymerization of thesilane groups together with a crosslinker (e.g., silane-PEG-silane, FIG.2, II), catalyzed by NaF, resulted in the proteins being wrapped in adegradable silica nanocapsules (NC), which efficiently protects theprotein from degradation in physiological conditions. Upon dissolutionof the silica NC and cleavage of the linker between the protein andsilane groups in physiological conditions, the protein is released toits original form (FIG. 4B).

Methods

The linker of Formula I (109 μg, 50 equiv. of protein) dissolved in 21.8μL DMSO was added to IL-2-fc (500 μg) solution in 478 μL PBS buffer. Themixture was rotated at 4° C. for 3 hours. Modified IL-2-fc was washedwith PBS (15 mL×3) using a Millipore Amicon ultra-centrifugal filter(molecular weight cutoff=10,000 Da). Purified IL-2-fc-linker conjugatein 500 μL PBS was mixed with (3-aminopropyl) triethoxysilane (55.2 μg,50 equiv. of protein) and rotated at 4° C. for 3 hours. The resultantsilane functionalized IL-2-fc was washed with PBS (15 mL×3) usingMillipore Amicon ultra-centrifugal filter to remove unreacted smallmolecules. IL2-fc-silane dissolved in 500 μL was then mixed withsilane-PEG-silane (100 μg, FIG. 2, II) followed by the addition ofsodium fluoride (200 μg). The mixture was stirred at 4° C. overnight.The resultant IL-2-fc-silica NCs were washed with PBS (15 mL×3) usingultra-centrifugal filter. The incorporation efficiency of IL-2-fc wasdetermined by centrifuging down the NCs and measuring the concentrationof IL-2 in the supernatant using ELISA kit. The loading of IL-2-fc inthe final IL-2-fc-NC was calculated based on the incorporationefficiency of IL-2-fc and assuming all the silane-PEG-silane is reactedand in the final NCs.

The successful conjugation of silane groups to IL-2 was demonstrated bymatrix-assisted laser desorption/ionization (MALDI) analysis (FIG. 3B).The degree of modification of IL-2 can be measured by calculating theincreased molecular weight. The results indicate that about 16 lysineresidues of IL-2 were covalently attached with silane groups (FIG. 3B).The prepared IL-2-silica NCs was 222.5±5.2 nm in diameter, as shown inboth dynamic light scattering (DLS) and scanning electron microscope(SEM) characterization (FIGS. 3C and 3D). Extraordinarily highincorporation efficiency (e.g., 95.5%) and high protein drug loading(e.g., 84.0%) was achieved using this reversible covalent modificationmethod (FIG. 4A). By comparison, an encapsulation method of the priorart typically results in less than 10% incorporation efficiency and ˜1%drug loading.

The triggered release of IL-2 from the IL-2-silica NCs was verified byincubating IL-2-silica NCs in buffer of different pH at 37° C. andanalyzing the release kinetics of IL-2 with or without reductantreagent, dithiothreitol (DTT). At pH 7.4, addition of 10 mM DTT resultedin 2.6 times faster release of IL-2 over 48 h incubation relative torelease without reductant reagent, demonstrating the redox responsiverelease of IL-2 (FIG. 4C). When the NCs were incubated in buffer with apH of 9.0, which facilitates the degradation of the silica shell, therelease of IL-2 was accelerated (FIG. 4C). These findings demonstratethat IL-2 was effectively conjugated to a reductant cleavable bond andprotected by a silica shell.

To stimulate the adoptive transferred T cells specifically andefficiently, IL-2-silica NCs were conjugated directly onto the plasmamembrane of donor cells, enabling continuous pseudoautocrine stimulationof transferred cells in vivo (FIG. 1). The silica NCs prevent thedegradation of IL-2 by proteases and allow for sustained local releaseof IL-2 in physiological conditions to expand cytotoxic T cellsspecifically without activating bystander T cells or expanding Tregs,thus avoiding the serious systemic toxicity of high-dose IL-2.IL-2-silica NCs were first functionalized with maleimide groups usingsilane chemistry (FIG. 2, III). The maleimide functionalized IL-2-silicaNCs were then covalently attached to the surface of adoptive transferredT cells through a maleimide-thiol reaction (FIG. 5A). Residual maleimidegroups of the NCs were quenched by in situ conjugation ofthiol-terminated polyethylene glycol (PEG-SH, Mw=5 kDa) (FIG. 5A). Thesuccessful cell surface conjugation was evidenced by flow cytometryanalysis of the T cells with surface bound fluorescence dye labeledIL-2-silica NCs (FIG. 5A).

To evaluate whether the surface bond IL-2-silica NCs could release IL-2with retained biological activity and expand CD8⁺ T cells in vitro,purified CD8⁺ T cells from splenocytes of mice were treated with freeIL-2, or conjugated with IL-2-silica NCs of equivalent amount of IL-2,and then co-cultured with CD3CD28 beads of 1:1 ratio. Cell proliferationwas monitored by both manual counting and analyzing carboxyfluorescein(CFSE) dilution by flow cytometry (FIGS. 5C and 5D). At twoconcentrations tested (3.0 g/mL or 7.5 μg/mL), surface bond IL-2-silicaNCs induces the comparable level of T cells expansion with free IL-2until day 3.

To further test the potential functional impact of stimulatoryIL-2-silica NCs in vivo, the response of Pmel-1 melanoma specificT-cells was assessed in vivo during adoptive transfer treatment ofB16F10 tumors in a murine metastatic lung tumor model. B16F10 melanomacells were injected through the tail vein to allow lung metastases toestablish for 6 days. Animals were then lympho-depleted and receivedadoptive transfer of luciferase-expressing Pmel-1 melanoma-specific CD8⁺T-cells with no further treatment, free IL-2 or surface-conjugatedIL-2-silica NCs, respectively in each group. T-cell expansion wasfollowed over time by bioluminescence imaging. Adoptively-transferredcells, without further adjuvant support, showed a low level persistencein the tumor-bearing recipients, which gradually declined over 6 days,as expected in the absence of additional stimulation or protection fromtumor immunosuppression (FIG. 6B). To assess the relative potency ofstimulation achieved by surface-conjugated IL-2-silica NCs compared totraditional systemic IL-2 therapy, the expansion of T-cells followinginjection of soluble IL-2 was compared to the expansion of T-cells withsurface-conjugated IL-2-silica NCs (at an equivalent total amount ofIL-2). T cells with surface-conjugated IL-2-silica NCs expanded to ahigher level on day 4 and day 6 relative to the T-cells with solubleIL-2 injection (FIG. 6B). Flow cytometry analysis of T-cells pooled fromthe inguinal lymph nodes on day 6, after adoptive transfer, confirmedthat the frequency of tumor-specific CD8⁺ T-cells (pmel-1 T-cellsexpress Thy1.1) was nearly 5 times greater in mice that received T-cellswith surface-conjugated IL-2-silica NCs relative to T-cells with solubleIL-2 (FIG. 6B). Soluble IL-2 showed no enhancement in T-cell expansioncompared to the injection of T cells alone. Thus, surface-conjugatedIL-2-silica NCs resulted in enhanced and more sustained T-cellsexpansion in tumor bearing mice compared with soluble IL-2.

Example 2

Linker-1 was first conjugated to the end of a 4 arm-PEG polymer chain(FIG. 8A). A subsequent crosslinking reaction of 4 arm-PEG-Linker-1 andIL-2-fc in PBS buffer resulted in protein-PEG nanogel particle formation(FIG. 8B), which efficiently protects the protein from degradation inphysiological conditions. Upon reductant dependent cleavage of thelinker between the protein and PEG in physiological conditions, theprotein is released to its original form.

Methods

FIG. 8A: Synthesis of 4 arm-PEG-Linker-1. 4 arm-PEG-NH2 (10 mg)dissolved in 300 μL tetrahydrofuran (THF) was added drop-wise to an 800μL THF solution of Linker-1 (17.4 mg, 40 equiv.) and triethylamine (5μL). The mixture was further stirred at room temperature overnight.Four-arm-PEG-Linker-1 was purified by dialysis (molecular weightcutoff=3,000 Da).

FIG. 8B: IL2-fc-PEG nanogel particle formation. IL-2-fc (50 μg) wasmixed with 4 arm-PEG-Linker-1 (15 μg, 3 equiv.) in 100 μL PBS buffer androtated at 4° C. overnight. The resultant IL2-fc-PEG nanogel was washedwith PBS (15 mL×3) using Millipore Amicon ultra-centrifugal filter(molecular weight cutoff=100,000 Da) to remove unreacted IL-2-fc or 4arm-PEG-Linker-1. The incorporation efficiency of IL-2-fc was determinedby centrifuging down the nanogels and measuring the concentration ofIL-2-fc in the supernatant. The loading of IL-2-fc in the finalIL2-fc-PEG nanogel (NG) was calculated based on the incorporationefficiency of IL-2-fc and assuming all 4 arm-PEG-Linker-1 is reacted andin the final nanogels.

The formation of IL2-fc-PEG nanogel particles was demonstrated by DLSmeasurement. The as prepared IL2-fc-PEG nanogel was 226.8±8.4 nm indiameter.

Example 3

Preparation and Characterization of Protein Nanogel (NG)

As shown in FIG. 9, IL2-Fc (100 μg) was mixed with a disulfidecrosslinker (4.36 μg, 10 equiv.) in 10 μL of phosphate buffered saline(PBS) and incubated at room temperature for 1 hour (h). The resultantIL2-Fc-crosslinked nanogel was washed with PBS (0.4 mL×3) usingMillipore Amicon ultra-centrifugal filter (molecular weightcutoff=100,000 Da) to remove unreacted IL2-Fc and/or unreacted disulfidecrosslinker. The IL2-Fc-crosslinked nanogel was then PEGylated by mixingthe IL2-Fc-crosslinked nanogel with 4 arm-PEG10k-NH2 (FIG. 9A; 50 μg, 5equiv.) in 100 μL PBS buffer. The mixture was incubated at roomtemperature for 0.5 h. The PEGylated IL2-Fc-crosslinked nanogel was thenwashed with the ultra-centrifugal filter. The incorporation efficiencyof IL2-Fc was determined by centrifuging down the PEGylatedIL2-Fc-crosslinked nanogels and measuring the concentration of IL2-Fc inthe supernatant.

The PEGylated IL2-Fc-crosslinked nanogel was analyzed with HPLC equippedwith a size exclusion column (FIG. 10A). By comparison with free IL2-Fc,the shift of the peak indicates the formation of crosslinked proteinwith much a larger molecular weight. The PEGylated IL2-Fc-crosslinkednanogel was further analyzed with transmission electron microscopy (TEM;FIG. 10B) and dynamic light scattering (DLS; FIG. 10C) to characterizethe size and morphology of the nanogels. The TEM image in FIG. 10B showsthat the size of PEGylated IL2-Fc-crosslinked nanogel as a dry solid is˜50-60 nm in diameter. The DLS characterization of the PEGylatedIL2-Fc-crosslinked nanogel in PBS solution indicated that thehydrodynamic size of nanogel is ˜85.6 nm. The larger hydrodynamic sizerelative to the size as a dry solid is due to the hydration of surfacePEG on the PEGylated IL2-Fc-crosslinked nanogel.

The PEGylated IL2-Fc-crosslinked nanogel can release intact IL2-Fc inphysiological condition. FIG. 11A, without being bound by, schematizes arelease mechanism of the IL2-Fc from NG without any chemical residuesremaining on the protein molecule. FIG. 11B shows controlled sustainedrelease of IL2-Fc in complete Roswell Park Memorial Institute (RPMI)media (media for T cells in vitro). When glutathione (GSH), a reducingagent in physiological condition, was added, the release kinetics wasaccelerated. The released mixture was also characterized with HPLC asshown in FIG. 11C: the peak of crosslinked IL2-Fc NG decreased, whilethe peak for free IL2-Fc increased over time. The released IL2-Fc wasalso characterized with matrix-assisted laser desorption/ionization(MALDI) mass spectroscopy to demonstrate that the released IL2-Fc hasthe same molecular weight as the native IL2-Fc, indicating that nochemical residue remained the IL2-Fc molecule.

Similar protein nanogels can be formulated using therapeutic proteinsother than IL-2-Fc. For example, human IL-15 superagonist(hIL-15Sa)-crosslinked and native mouse IL-2 (mIL-2)-crosslinkednanogels are represented by HPLC curves in FIG. 12.

At protein concentrations of greater than 50 mg/mL, bulk gel formedinstead of nanogel (FIG. 13).

Example 4

Carrier Free Delivery of a Cytokine Using a Protein Nanogel to Augment TCells for Adoptive Cell Therapy for Cancer

To demonstrate the application of a protein nanogel of the presentdisclosure, in the context of adoptive T cell transfer (ACT) for cancerimmunotherapy, a IL2-Fc-crosslinked nanogel was used to deliver IL2-Fcto specifically expand adoptive transferred T cells in vivo. AnIL2-Fc-crosslinked nanogel provides a highly efficient way to deliver asufficient amount of the cytokine to the T cell surface throughconjugation. An IL2-Fc-crosslinked nanogel was surface modified with adisulfide crosslinker (Nat. Med. 16, 1035-1041, 2010, incorporated byreference herein; FIG. 9) and then conjugated to the surface of effectorT cells. As shown in FIG. 14A, the IL2-Fc-crosslinked nanogel can beconjugated to the surface of T cells. By controlling the amount ofIL2-Fc-crosslinked nanogels added to the T cells, the T cell surfacedensity could be well controlled, as evidenced by flow cytometryanalysis using fluorescently-labeled IL2-Fc-crosslinked nanogels (FIG.14B).

The Pmel-1 melanoma-specific CD8⁺ T-cells with surface-conjugatedIL2-Fc-crosslinked nanogels was adoptive transferred to mice withestablished lung metastases of B16F10 melanoma. The in vivo expansion ofthe transferred T cells was monitored over time using bioluminescenceimaging. The CD8⁺ T-cells with surface-conjugated IL2-Fc-crosslinkednanogels showed markedly increased in vivo expansion relative to Tcell-only controls or T cells with systemically administered free IL2-Fc(FIGS. 15A-15B).

The frequency of adoptively-transferred T cells and endogenous T cellsin the inguinal lymph nodes and blood were analyzed with flow cytometryon Day 12 after adoptive transfer. By comparing with the group ofPmel-1⁺ systemic IL2-Fc, the T cells with conjugated IL2-Fc-crosslinkednanogels showed 4.8 and 2.0 fold increased frequency of transferred CD8⁺T-cells in LN and blood respectively. However, the systemic IL2-Fcexpanded the endogenous CD8⁺ T-cells nonspecifically in both theinguinal lymph nodes and blood (FIG. 16).

To evaluate the efficacy of the treatment of adoptive cell transfer, allthe lungs were collected. By counting the number of tumor nodules inlung, it was shown that the mice treated with T cells having conjugatedIL2-Fc-crosslinked nanogels had the lowest number of tumors (FIGS.17A-17B), indicating that the specific expansion ofadoptively-transferred T cells by conjugated IL2-Fc-crosslinked nanogelsresulted in improved efficacy against the lung metastases of B16F10melanoma. The efficacy results were further confirmed with histologicalanalyses, and the mice treated with T cells with conjugatedIL2-Fc-crosslinked nanogels also had the lowest grade of lung tumorburden (FIGS. 17C-17D).

T cell surface bound IL2-Fc-crosslinked nanogels provided long-lasting,specific expansion of adoptively-transferred T cells through sustainedrelease of intact IL2-Fc in vivo and, thus, improved the efficacy ofadoptive T cell therapy against cancer.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements).

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements).

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

All references (e.g., published journal articles, books, etc.), patentsand patent applications disclosed herein are incorporated by referencewith respect to the subject matter for which each is cited, which, insome cases, may encompass the entirety of the document.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method for treating cancer by stimulating orenhancing a tumor antigen-specific immune response in a human subject,the method comprising: administering to the human subject a compositioncomprising (i) an expanded population of isolated T cells that isspecific to more than one tumor antigen, wherein the T cells are notgenetically engineered; and (ii) a nanostructure conjugated to thesurface of each T cells, the nanostructure comprising a plurality ofbiologically-active proteins reversibly and covalently crosslinked toeach other through a degradable linker, wherein the biologically-activeproteins comprise an immunostimulatory cytokine, and wherein the linkerdegrades under physiological conditions to release thebiologically-active proteins, wherein upon release in vivo, thebiologically-active proteins stimulate or enhance a tumorantigen-specific response in a human subject, thereby treating cancer.2. The method according to claim 1, wherein the immunostimulatorycytokine is an IL-2, IL-7, IL-15, IL-15 superagonist, IL-12, IFN-gamma,IFN-alpha, GM-CSF, or FLT3-ligand.
 3. The method according to claim 1,wherein the immunostimulatory cytokine is an IL-15 or an IL-15superagonist.
 4. The method according to claim 1, wherein thebiologically-active protein is a fusion protein.
 5. The method accordingto claim 1, wherein the T cells comprise CD4+ T cells, CD8+ T cells,cytotoxic T cells, or NK T cells.
 6. The method according to claim 1,wherein the T cells are autologous to the human subject.
 7. The methodaccording to claim 1, wherein the nanostructure is noncovalentlyconjugated to the surface of each T cell.
 8. An immunostimulatorycomposition for treating cancer by stimulating or enhancing a tumorantigen-specific immune response in a human subject, the compositioncomprising (i) an expanded population of isolated T cells that isspecific to more than one tumor antigen, wherein the T cells are notgenetically engineered; and (ii) a nanostructure conjugated to thesurface of each T cell, the nanostructure comprising a plurality ofbiologically-active proteins reversibly and covalently crosslinked toeach other through a degradable linker, wherein the biologically-activeproteins comprise an immunostimulatory cytokine, and wherein the linkerdegrades under physiological conditions to release thebiologically-active proteins, wherein upon release in vivo, thebiologically-active proteins stimulate or enhance a tumorantigen-specific response in a human subject, thereby to treat cancer.9. The composition according to claim 8, wherein the immunostimulatorycytokine is an IL-2, IL-7, IL-15, IL-15 superagonist, IL-12, IFN-gamma,IFN-alpha, GM-CSF, or FLT3-ligand.
 10. The composition according toclaim 8, wherein the immunostimulatory cytokine is an IL-15 or an IL-15superagonist.
 11. The composition according to claim 8, wherein thebiologically-active protein is a fusion protein.
 12. The compositionaccording to claim 11, wherein the fusion protein comprises an Fc. 13.The composition according to claim 11, wherein the immunostimulatorycytokine is an IL-15 or an IL-15 superagonist.
 14. The compositionaccording to claim 8, wherein the T cells comprise CD4+ T cells, CD8+ Tcells, cytotoxic T cells, or NK T cells.
 15. The composition accordingto claim 8, wherein each T cell is conjugated to a plurality ofnanostructures.
 16. The composition according to claim 8, wherein thenanostructure is noncovalently conjugated to the surface of each T cell.17. The composition according to claim 8, wherein the T cells compriseCD8+ T cells, the immunostimulatory cytokine is an IL-15 or IL-15superagonist, and the nanostructure is noncovalently conjugated to the Tcells.
 18. The composition according to claim 8, wherein thenanostructure further comprises a polymer.
 19. The composition accordingto claim 18, wherein the polymer comprises poly(ethylene oxide),polylactic acid, poly(lactic-co-glycolic acid), polyethylene glycol,polyglutamate, or polylysine.
 20. The composition according to claim 18,wherein the polymer comprises polylysine.